Background <p>Glioma remains a highly aggressive malignancy with limited treatment efficacy. Kaempferitrin (KM), a natural flavonoid glycoside, has been attracted attention for its potential anti-cancer activity. This study specifically explored the effect of KM on glioma progression and the involved underlying mechanism.</p> Methods <p>Cell viability, apoptosis, invasion, migration, and cancer stem cell properties were evaluated via CCK-8 assays, flow cytometry assays, transwell invasion assays, wound-healing assays, and sphere formation assays, respectively. Flow cytometry was also employed to quantify CD206-positive cells. Protein expression of C-X-C motif chemokine receptor 2 (CXCR2) and ubiquitin specific peptidase 8 (USP8) was determined by western blotting. CXCR2 mRNA levels were detected using qRT-PCR. The interaction between USP8 and CXCR2 was investigated using co-immunoprecipitation (Co-IP), cycloheximide (CHX) chase, and ubiquitination assays. Xenograft mouse model, HE staining, and IHC assay was used to assess the effect of KM on tumor formation.</p> Results <p>KM treatment significantly induced apoptosis and suppressed key malignant phenotypes in glioma cells, including invasion, migration, cancer stem-like properties, and M2 macrophage polarization. Notably, KM downregulated the expression of both CXCR2 and USP8. CXCR2 overexpression counteracted the tumor-suppressive effects of KM. Mechanistically, USP8 stabilizes the CXCR2 protein through deubiquitination. Accordingly, overexpression of CXCR2 was sufficient to rescue the malignant phenotypes suppressed by USP8 knockdown, and USP8 overexpression abated KM-mediated inhibition of cell biological malignant behaviors. Finally, the anti-tumor efficacy of KM was confirmed in mice.</p> Conclusion <p>KM inhibited the malignant progression of glioma through inactivating the USP8/CXCR2 axis. These findings not only identify KM as a promising candidate but also underscore the USP8/CXCR2 signaling pathway as a potential target for glioma treatment.</p>